RESUMEN
BACKGROUND: Novel male-based contraceptives are needed to broaden family planning choices. A progestin, Nestorone® (Nes) gel, plus a testosterone (T) gel suppresses sperm concentrations to levels associated with effective contraception in normal men. However, administration of two gels on different parts of the body daily is impractical. OBJECTIVE: Compare the effectiveness of daily application of a single, combined 8.3 mg Nes-62.5 mg T gel (Nes-T) vs. 62.7 mg T gel to suppress serum FSH and LH concentrations to ≤1.0 IU/L (a threshold associated with suppression of sperm concentrations to ≤1 million and effective contraception) and to compare the pharmacokinetics of serum Nes and T concentrations between the gel groups. DESIGN: We conducted a 28-day, double-blind, controlled trial of 44 healthy men randomized to daily Nes-T or T gel with measurement of hormones at baseline, treatment, and recovery and during 24-h pharmacokinetic studies on days 1 and 28 of treatment. RESULTS: Of the subjects who met pre-defined inclusion criteria, 84% of the Nes-T group suppressed serum gonadotropin concentrations to ≤1.0 IU/L at days 21-28 vs. 16.7% in the T group (p < 0.001). On day 1, Nes concentrations rose significantly above baseline by 2 h and continued to rise up to 24 h after Nes-T gel application. Nes concentrations were not detectable in the T group. Serum total T concentrations rose and were significantly higher in the T gel group compared to the Nes-T group at 24 h on day 1 and days 11, 14, and 21 (p < 0.01). There were no serious adverse events in either group. About 80% of the subjects reported satisfaction with both gels. CONCLUSION: Daily Nes-T gel effectively and safely suppresses serum gonadotropins and is acceptable to most men. It should be studied further in efficacy trials of hormonal male contraception.
Asunto(s)
Agentes Anticonceptivos Hormonales/farmacología , Anticonceptivos Masculinos/farmacología , Gonadotropinas/sangre , Norprogesteronas/farmacología , Testosterona/farmacología , Adolescente , Adulto , Agentes Anticonceptivos Hormonales/farmacocinética , Anticonceptivos Masculinos/farmacocinética , Método Doble Ciego , Combinación de Medicamentos , Hormona Folículo Estimulante/sangre , Anticoncepción Hormonal , Humanos , Hormona Luteinizante/sangre , Masculino , Persona de Mediana Edad , Norprogesteronas/farmacocinética , Recuento de Espermatozoides , Espermatogénesis/efectos de los fármacos , Encuestas y Cuestionarios , Testosterona/farmacocinética , Congéneres de la Testosterona/farmacología , Adulto JovenRESUMEN
BACKGROUND: Testosterone (T)/Nestorone (NES) combination gel is a potential transdermal male contraceptive that suppresses gonadotropins and spermatogenesis. Transfer of transdermal T from men to women can be prevented by washing or covering application sites with clothing. OBJECTIVES: We hypothesized that showering or wearing a shirt over gel application sites would prevent secondary exposure of T and NES to a woman after close skin contact. MATERIALS AND METHODS: Twelve healthy male and 12 healthy female participants were recruited. Men applied T/NES 62 mg/8 mg gel to their shoulders and upper arms. Two hours after application, female partners rubbed the application site for 15 min. Exposure in the female partner was assessed under three conditions: a shirt covered the application site; the man showered prior to skin contact; or without intervention to reduce transfer. Serum T and NES concentrations were measured by LC-MS/MS in serial blood samples for 24 h after gel exposure. MAIN OUTCOMES: Change in female serum T and NES levels as measured by average concentration over 24 h (Cavg ). RESULTS: Median female serum T Cavg was 23.9 ng/dL (interquartile range, 19.3, 33.9) with the shirt barrier and 26.7 ng/dL (20.7, 33.9) after showering, which was higher than baseline 20.9 ng/dL (16.7, 25.0), both p < 0.03) but lower than without intervention (58.2 ng/dL [30.9, 89.1], both p < 0.01). Female serum NES Cavg and maximum concentration were below the lower limit of quantification with the shirt barrier and after showering, but increased without intervention in six of 12 women (maximum concentration <60 pg/mL). Men had lower average serum NES levels after showering (47 pg/ml [20, 94] compared to no intervention (153.3 pg/mL [51, 241], p < 0.02). CONCLUSION: Secondary transfer of T and NES occurs after intensive skin contact with the gel application site. Secondary transfer is decreased by a shirt barrier or showering before contact.
Asunto(s)
Anticonceptivos Masculinos/administración & dosificación , Anticonceptivos Masculinos/farmacocinética , Norprogesteronas/administración & dosificación , Norprogesteronas/farmacocinética , Testosterona/administración & dosificación , Testosterona/farmacocinética , Adulto , Femenino , Geles , Humanos , Masculino , PielRESUMEN
BACKGROUND: As part of a program to develop a novel estradiol-releasing contraceptive vaginal ring (CVR), we evaluated the pharmacokinetic (PK) profile of CVRs releasing segesterone acetate (Nestorone® (NES)) combined with one of three different estradiol (E2) doses. STUDY DESIGN: A prospective, double-blind, randomized, multi-centered study to evaluate a 90-day CVR releasing NES [200mcg/day] plus E2, either 10mcg/day, 20mcg/day, or 40mcg/day in healthy reproductive-age women with regular cycles. Participants provided blood samples twice weekly for NES and E2 levels during the first 60 days (ring 1) and the last 30 days (ring 2) of use. A subset underwent formal PK assessments at ring initiation, ring exchange (limited PK), and study completion. RESULTS: The main study enrolled 197 women; 22 participated in the PK substudy. Baseline characteristics between the main and PK participants were comparable, with an average BMI of 25.8 kg/m2 (SD 4.3). In the PK substudy, all three rings showed similar NES PK: mean area under the curve (AUC(0-72)) 34,181 pg*day/mL; concentration maximum (Cmax) 918 pg/mL; time to maximum concentration (Tmax) 3.5 h. For E2, the Cmax occurred at 2 h, and was significantly higher with the 20 mcg/day ring (mean 390 pg/mL); 10 mcg/day, 189 pg/mL, p=.003; 40 mcg/day, 189 pg/mL, p<.001), and declined rapidly to≤50 pg/mL for all doses by 24 h. For all subjects, the median E2 levels remained under 35 pg/mL during treatment. CONCLUSION: PK parameters of NES were not affected when paired with different doses of E2, but E2 levels from all three doses were lower than anticipated and no dose response was observed. IMPLICATIONS: While these novel estradiol-releasing combination contraceptive vaginal rings provided sustained release of contraceptive levels of Nestorone over 90 days, the E2 levels achieved were not consistent with bone protection, and a dose-response was not observed.
Asunto(s)
Anticonceptivos Femeninos/farmacocinética , Dispositivos Anticonceptivos Femeninos , Estradiol/farmacocinética , Norprogesteronas/farmacocinética , Adulto , Anticoncepción , Anticonceptivos Femeninos/administración & dosificación , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Estradiol/administración & dosificación , Femenino , Humanos , Norprogesteronas/administración & dosificación , Estudios Prospectivos , Estados Unidos , Adulto JovenAsunto(s)
Hormona Liberadora de Gonadotropina/antagonistas & inhibidores , Antagonistas de Hormonas/uso terapéutico , Anticonceptivos Masculinos , Femenino , Enfermedades de los Genitales Femeninos/tratamiento farmacológico , Neoplasias de los Genitales Femeninos/tratamiento farmacológico , Hormona Liberadora de Gonadotropina/análogos & derivados , Hormona Liberadora de Gonadotropina/uso terapéutico , Humanos , Masculino , Neoplasias de la Próstata/tratamiento farmacológico , Técnicas ReproductivasRESUMEN
Human chorionic gonadotropin (hCG) preparations contain activity against HIV type 1 (HIV-1). However, there has been controversy about whether some biological activities of hCG beta-subunit (hCGbeta) preparations are caused by the beta-subunit itself or other proteins present in the preparations. We report here the purification, characterization, and identification of three enzymes with anti-HIV activity present in the beta-core fraction of hCGbeta prepared from the urine of pregnant women. The N-terminal amino acid sequence of one protein is identical to human urinary lysozyme C, and those of the other two are identical to human RNase A and urinary RNase U. We thus refer to these proteins as AVL (antiviral lysozyme) and AVR (antiviral RNases). In addition to HIV-1 inhibition, AVL is capable of lysing Micrococcus lysodeikticus. AVR digests a variety of RNA substrates, including RNA from HIV-1-infected cells. We also find that lysozyme from chicken egg white, human milk, and human neutrophils and RNase A from bovine pancreas possess activity against HIV-1. These findings may offer additional strategies for the treatment of HIV-1 infection.
Asunto(s)
Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Gonadotropina Coriónica/química , Gonadotropina Coriónica/farmacología , VIH-1/efectos de los fármacos , Muramidasa/farmacología , Ribonucleasas/farmacología , Secuencia de Aminoácidos , Animales , Fármacos Anti-VIH/aislamiento & purificación , Bovinos , Femenino , Humanos , Datos de Secuencia Molecular , Muramidasa/química , Muramidasa/aislamiento & purificación , Embarazo , Ribonucleasas/química , Ribonucleasas/aislamiento & purificaciónRESUMEN
Glycoprotein hormone alpha subunit, in its free form (free alpha), is a major placental product. Its glycosylation was found to change dramatically during the advancement of pregnancy. In this study, we have analyzed these glycosylation changes in five normal pregnancies. Binding to Lens culinaris lectin increased dramatically in all subjects between weeks 14 and 17 from the last menstrual period, indicating more core fucosylation as well as possible changes in branching of glycans. Studies using Datura stramonium agglutinin confirmed that the type of triantennary branching changed in this period of pregnancy. The precise structural nature of these changes was determined by high-pH anion-exchange chromatography and electrospray ionization mass spectrometry. Amounts of core fucosylation and of triantennary glycans increased substantially from early to late second trimester, and a shift was observed from 1-->4/1-->3- toward predominantly 1-->6/1-->6-branched triantennary structures. The glycosylation changes occurred in all five individuals at the same time period in gestation, suggesting developmental regulation of N-acetylglucosaminyltransferases IV and V and alpha6-fucosyltransferase during normal pregnancy. These enzymatic activities also appear to be affected in malignant transformation of the trophoblast. Our findings have important implications for the proposed use of specific forms of glycosylation as markers for cancer, as the relative amounts of these glycans in normal pregnancy will be determined by gestational age.
Asunto(s)
Hormonas Glicoproteicas de Subunidad alfa/orina , Secuencia de Carbohidratos , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Femenino , Hormonas Glicoproteicas de Subunidad alfa/química , Hormonas Glicoproteicas de Subunidad alfa/aislamiento & purificación , Glicosilación , Humanos , Lectinas/química , Espectrometría de Masas , Datos de Secuencia Molecular , Polisacáridos/química , EmbarazoRESUMEN
During the secretory phase of the menstrual cycle, endometrial stromal cells differentiate into decidual cells, which play a crucial role in implantation and maintenance of pregnancy. In this and our previous study, we demonstrate that glycoprotein hormone free alpha-subunit potentiates progesterone-mediated decidualization of human endometrial stromal cells in vitro. Although addition of intact hCG to cultures resulted in stimulatory activity, its potency was 20-fold less than that of alpha-subunit. However, in the present study we show that decidualizing endometrial cells actively generate uncombined alpha-subunit by dissociating hCG. The amount of dissociated alpha-subunit could fully account for the stimulatory activity observed with hCG. Active dissociation of hCG was dependent on the presence of endometrial cells and did not occur in conditioned medium, excluding involvement of a stable secreted factor such as a protease. In addition to dissociated alpha- and beta-subunits, minor amounts of beta-core and alpha-fragments were detected as degradation products during active dissociation. We also observed an increase in beta-immunoreactivity that coeluted with hCG on size-exclusion gel chromatography, indicating that a portion of the still dimeric hCG may have been nicked in the dissociation process. However, using an assay with specificity for nicked hCG, we showed that dissociation of hCG was not produced from a pool of preexisting nicked hCG. These findings more firmly establish the concept that gonadotropin hormone free alpha-subunit plays a role in the regulation of human endometrial cell differentiation. In addition, identification of the various products formed by incubation of hCG with decidualizing cells yielded insight into the mechanism of hCG degradation, and may explain some activity previously ascribed to hCG.
Asunto(s)
Decidua/fisiología , Endometrio/metabolismo , Hormonas Glicoproteicas de Subunidad alfa/metabolismo , Hormonas Glicoproteicas de Subunidad alfa/farmacología , Progesterona/farmacología , Células del Estroma/metabolismo , Adulto , Células Cultivadas , Gonadotropina Coriónica/farmacología , Medios de Cultivo Condicionados , Decidua/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Humanos , Cinética , Persona de Mediana Edad , Ovulación , Prolactina/biosíntesisRESUMEN
Highly purified human chorionic gonadotropin (hCG) interacts with the thyrotropin (TSH) receptor and stimulates triiodothyronine (T3) secretion, iodide uptake and organification, and cyclic adenosine monophosphate (cAMP) formation in human thyroid follicles. Because of interest in the role of the carbohydrate component in the structure-function relationships of hCG we undertook to deplete hCG of its sialic acid or carbohydrate residues and assess the thyrotropic activity of the carbohydrate-modified forms. For this purpose, we used our assay system consisting of human thyroid follicles cultured and suspended in collagen gel in serum-free medium. Under these conditions, the cells are organized as follicular three-dimensional structures with normal polarity, enabling enhanced responsiveness to hormonal stimulation, and T3 secretion can be measured as a response parameter. Desialylated (ds)-hCG and deglycosylated (dg)-hCG dose-dependently stimulated T3 secretion, iodide uptake and organification, and in each case did so with about twice the intrinsic activity of native hCG. Indeed, removal of the sialic acid or carbohydrate residues from native hCG transformed it into a thyroid stimulator that elicited a maximal response in terms of iodide uptake, organification and T3 secretion by human thyroid follicles as high as TSH and almost twice as high as native hCG. Not only were ds-hCG and dg-hCG more intrinsically active than hCG, they were more than five times as potent. As with hCG, both ds-hCG and dg-hCG managed to elicit such responses in human thyrocytes while evoking minimal amounts of cAMP, illustrating the concept of cAMP superfluity and highlighting the potential pitfalls of using cAMP as a measure of hormonal bioactivity. hCG, and to a greater extent ds-hCG and dg-hCG, inhibited, as did TSH, gamma-interferon-induced human leukocyte antigen-DR (HLA-DR) expression in human thyrocytes, again reflecting the intrinsic thyrotropic activity of native hCG and its variants depleted of sialic acid or carbohydrate residues. In conclusion, this is the first report on the thyrotropic activity of ds-hCG and dg-hCG using the physiologically relevant hormonal end-point response, thyroid hormone secretion. The study was conducted in a serum-free culture system of human thyroid follicles and shows that removal of the sialic acid or carbohydrate residues from native hCG transform hCG variants into thyroid stimulating superagonists. The hCG variants inhibited, as did TSH, gamma-interferon-induced HLA-DR expression.
Asunto(s)
Asialoglicoproteínas/farmacología , Gonadotropina Coriónica/agonistas , Gonadotropina Coriónica/farmacología , Glándula Tiroides/efectos de los fármacos , Células Cultivadas , AMP Cíclico/metabolismo , Antígenos HLA-DR/inmunología , Humanos , Yoduros/metabolismo , Pruebas de Función de la Tiroides , Glándula Tiroides/citología , Glándula Tiroides/inmunología , Glándula Tiroides/fisiología , Triyodotironina/metabolismoRESUMEN
Glycoprotein hormone-free alpha subunit is secreted by the pituitary throughout the menstrual cycle and by the placenta during pregnancy. We showed previously that free alpha subunit stimulated PRL secretion from term pregnancy decidual cells, suggesting a function for free alpha in pregnancy. However, no role has been ascribed to free alpha in the normal menstrual cycle. Using an in vitro model, we examined the role of alpha subunit in regulating human endometrial stromal cell differentiation (decidualization). PRL and insulin-like growth factor binding protein-1 (IGFBP-1), specific decidual secretory products, were used as markers of decidualization. We found that alpha subunit acted synergistically with progesterone (P) to induce more rapid decidualization with higher output (2- to 6-fold) of PRL and IGFBP-1, compared with P alone (P < 0.01). The effect of alpha was dose dependent, with stimulatory activity starting at 0.05 ng/ml and reaching maximal levels at 1-2 ng/ml. These levels correspond to serum concentrations of free alpha found during the luteal phase of the menstrual cycle when endometrial decidualization occurs in vivo. These findings demonstrate new biological activity for alpha subunit in the regulation of human endometrial decidualization and indicate that free alpha plays a role in human reproduction. Furthermore, demonstration of potential bioactivities of free alpha subunit has important implications for understanding normal endocrine function and various pathological conditions.
Asunto(s)
Decidua/citología , Endometrio/citología , Hormonas Glicoproteicas de Subunidad alfa/farmacología , Progesterona/farmacología , Células del Estroma/citología , Adulto , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Gonadotropina Coriónica/farmacología , Sinergismo Farmacológico , Endometrio/metabolismo , Femenino , Hormonas Glicoproteicas de Subunidad alfa/fisiología , Humanos , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Persona de Mediana Edad , Embarazo , Prolactina/biosíntesis , Reproducción/fisiologíaRESUMEN
The extraembryonic coelomic fluid (EECF) represents a major compartment in the fetal-placental unit during the first trimester of pregnancy. The compartment is composed of the fluid contained between the chorionic and amniotic membranes. The levels of glycoprotein hormone free alpha-subunit and free beta-subunit in the EECF far exceed those in the amniotic fluid or maternal serum. Furthermore, the level of free alpha in this compartment is twice that of intact hCG. We purified the glycoprotein hormone free alpha-subunit from a pool of EECF. This free alpha-subunit was found to be larger in size than the alpha-subunit of intact hCG. The size difference was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reduced and denatured conditions. The carbohydrate composition of the EECF free alpha-subunit indicated a higher degree of oligosaccharide branching, as evidenced by larger amounts of fucose, sialic acid, galactose, and N-acetylglucosamine than were present on combined hCG alpha. These differences in size and carbohydrate composition argue strongly against the concept that free alpha-subunits might originate from dissociation of intact hCG or "nicked" hCG. The free subunits of the EECF were evaluated for their ability to combine with the corresponding subunit obtained by dissociation of intact hCG. EECF free beta was able to combine with hCG alpha to form intact hCG. In contrast, EECF free alpha was unable to combine with hCG beta to form intact hCG. However, after removal of the asparagine-linked glycans by treatment with N-glycanase, most of the previously uncombinable free alpha-subunits were able to combine with hCG beta. These data demonstrate that the N-linked oligosaccharide(s) of EECF free alpha function to prevent the molecule from combining with the available and combinable free beta-sub-units that coexist in the same physiological compartment during early pregnancy. In view of the large amount of free alpha that is present in the EECF and the observation that, in vitro, free alpha can stimulate uterine decidual cell PRL secretion, together with the close apposition of free alpha-producing cells to decidual cells, it is likely that EECF free alpha has a function in early pregnancy. Carbohydrate modifications generated during the biosynthesis of EECF free alpha-subunit ensure that a population of free alpha molecules can exist in the presence of substantial quantities of free beta-subunits, and correspondingly, these same carbohydrate modifications function to permit the existence of free beta-subunits in the same gestational compartment with free alpha molecules.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Líquido Amniótico/metabolismo , Hormonas Glicoproteicas de Subunidad alfa/metabolismo , Primer Trimestre del Embarazo , Embarazo/metabolismo , Carbohidratos/análisis , Gonadotropina Coriónica/química , Gonadotropina Coriónica/aislamiento & purificación , Femenino , Hormonas Glicoproteicas de Subunidad alfa/química , Glicosilación , HumanosRESUMEN
Chorionic gonadotrophin and pituitary luteinizing hormone, follicle-stimulating hormone and thyroid-stimulating hormone comprise the family of glycoprotein hormones, which regulate major metabolic and reproductive functions of the body. These are heterodimeric glycoproteins composed of a common alpha subunit and a hormone-specific beta subunit. The N-linked oligosaccharides of these hormones are necessary for proper folding, assembly, secretion, metabolic clearance and biological activity. The free alpha subunit, which is shown to have a physiological function, is maintained in the uncombined form due to its glycan structures. The N-glycans of the glycoprotein hormones contain a variety of terminal residues, which are responsible for the differential targeting and clearance of the hormones. Glycosylation of these hormones is regulated by a variety of physiological and pathological conditions, leading to subtle alterations in their bioactivities. Recent studies on the structures and specific functions of different glycans of natural and recombinant glycoprotein hormones have provided valuable insight into the glycobiology of these hormones. This information will be useful in the development of diagnostic and therapeutic applications of the glycoprotein hormones.
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Hormonas Glicoproteicas de Subunidad alfa/química , Glicoproteínas/química , Gonadotropinas/química , Oligosacáridos/química , Tirotropina/química , Secuencia de Aminoácidos , Animales , Secuencia de Carbohidratos , Glicoproteínas/metabolismo , Glicosilación , Humanos , Tasa de Depuración Metabólica , Datos de Secuencia Molecular , Tirotropina/metabolismoRESUMEN
Despite extensive studies, the issue of whether hCG possesses intrinsic thyrotropic activity remains unresolved. This is mainly because in the experimental systems used so far, the parameters measured did not include the thyroid-specific functions of iodine organification and the hormonal end-point response, T3 secretion, and cells of nonhuman origin were employed, constituting a major drawback in view of the wide variation in sensitivity of thyroid responsiveness to hCG in different species. We investigated the thyrotropic activity of hCG, using for this purpose a novel homologous assay system consisting of human thyroid follicles cultured suspended in collagen gel in serum-free medium. Under these conditions, the cells are organized as follicular three-dimensional structures with normal polarity, enabling enhanced responsiveness to hormonal stimulation. The parameters measured were the thyroid-specific functions of iodide uptake, organification, and T3 secretion, as well as formation of the second messenger, cAMP. Purified hCG (biological potency, 21,700 IU/mg; with no detectable TSH by immunoradiometric TSH assay) did indeed exhibit thyroid stimulatory activity. At doses ranging from 10-400 mg/L, hCG induced a dose-dependent increase in the parameters measured. The rise from basal to maximal levels achieved after hCG stimulation was 1.3 to 3.6 pmol/well for cAMP formation, 34 to 21,408 cpm/well for iodide uptake, 261 to 20,167 cpm/well for iodide organification, and 40 to 927 fmol/well for T3 secretion. Maximal levels elicited by hCG (200 mg/L) relative to maximal values achieved with bovine TSH were 49%, 56%, and 42% for iodide uptake, organification, and T3 secretion, respectively, and only 5% for cAMP. Iodide uptake proved to be the most sensitive indicator of the thyrotropic activity of hCG, with increases occurring at a concentration of 10 mg/L. Acting as a partial agonist, hCG was also capable of dose-dependently inhibiting TSH-stimulated cAMP formation. The free alpha- and beta- subunits of hCG, at doses as high as 200 mg/L, had no thyroid-stimulating effect. The present data thus clearly demonstrate that hCG is a human thyroid stimulator. Moreover, hCG managed to elicit substantial biological cell responses in human thyrocytes while evoking minimal amounts of cAMP, illustrating the concept of cAMP superfluity and highlighting the potential pitfalls of using cAMP as a reliable measure of hormonal bioactivity.
Asunto(s)
Gonadotropina Coriónica/farmacología , AMP Cíclico/metabolismo , Yoduros/metabolismo , Glándula Tiroides/fisiología , Triyodotironina/metabolismo , Células Cultivadas , Gonadotropina Coriónica/administración & dosificación , Relación Dosis-Respuesta a Droga , Humanos , Radioisótopos de Yodo , Glándula Tiroides/efectos de los fármacos , Tirotropina/farmacologíaRESUMEN
Carbohydrate is important to the structure, function, and circulatory survival of the glycoprotein hormones. Human CG (hCG) and the related free alpha-molecule of pregnancy contain four and two asparagine-linked oligosaccharides, respectively. The present study analyzes changes in the glycosylation patterns of hCG and free alpha in early vs. late gestation. Five volunteers provided 24-h urine samples, weekly, throughout their pregnancies. Extracts of early pregnancy (weeks 7-12) and late pregnancy (weeks 28-32) urines were pooled. Early and late samples from each patient were subjected to gel filtration to separate hCG and free alpha, and the populations thus obtained were analyzed by lectin affinity chromatography on Concanavalin A-Sepharose (Con A) and Lens culinaris-agarose (Lch). Using Con A, free alpha and hCG were separated into an unbound fraction (eluted with Con A buffer), a weakly bound fraction (eluted with 10 mmol alpha-methyl-D-glucoside) and a tightly bound fraction (eluted with 500 mmol alpha-methyl-D-mannoside). For free alpha-molecule, a significant decrease in tightly bound Con A forms, was noted from early to late pregnancy with a mean difference of 17.0 +/- 2.4% (P less than 0.01). Concomitantly, in late pregnancy, an increase in Con A unbound forms of free alpha was noted with mean difference of 12.5 +/- 1.7% (P less than 0.01). These changes indicate the presence of more highly branched oligosaccharides on free alpha as gestation advances. No changes were noted in the Con A binding of intact hCG; nearly all hCG bound in both early and late pregnancy. Using Lch, free alpha and hCG were separated into an unbound fraction (eluted with Lch buffer) and a bound fraction (eluted with 500 mmol alpha-methyl-D-mannose). Both free alpha and intact hCG in late pregnancy exhibited increased binding to Lch, with mean differences from early to late pregnancy of 30.2 +/- 4.8% (P less than 0.01) and 11.4 +/- 4.5% (P less than 0.05), respectively. These data indicate increased incorporation of fucose into the carbohydrate moieties in late pregnancy. Taken together, these data derived by analysis using lectin specificity imply the presence of more highly branched, fucosylated oligosaccharides as gestation progresses.
Asunto(s)
Gonadotropina Coriónica/orina , Hormonas Glicoproteicas de Subunidad alfa/orina , Embarazo/orina , Gonadotropina Coriónica/metabolismo , Cromatografía de Afinidad , Concanavalina A , Dextranos , Femenino , Edad Gestacional , Hormonas Glicoproteicas de Subunidad alfa/metabolismo , Glicosilación , Humanos , Lectinas , Primer Trimestre del Embarazo , Tercer Trimestre del Embarazo , SefarosaRESUMEN
We have examined the carbohydrate composition of corticosteroid-binding globulin (CBG) obtained from rat and human serum. Rat CBG contained a carbohydrate composition that was strikingly different from that of human CBG. Like other glycoproteins that circulate in human plasma, human CBG had a carbohydrate composition that was consistent with the presence of biantennary and triantennary oligosaccharide structures. In contrast, the carbohydrate composition of rat CBG indicated the presence of more than one sialic acid residue per antenna. It is not clear whether rat CBG contains a carbohydrate structure with sialic acids attached to both galactose and N-acetylglucosamine on the same antenna, or a terminal disialylated structure (sialic acid linked alpha 2-8 to sialic acid). These structural variations may play a role in the interaction of CBG with its receptor.
Asunto(s)
Carbohidratos/análisis , Transcortina/química , Animales , Femenino , Glicosilación , Humanos , Embarazo , Ratas , Especificidad de la Especie , Transcortina/metabolismoRESUMEN
Free alpha molecules isolated from pregnancy, as well as highly purified reference preparations of hCG alpha subunit (CR119 or CR123), stimulated the release of prolactin from human decidual cells in culture. The amount of prolactin secreted during a 24 h incubation was concentration-dependent over a range of increasing doses of alpha from 0.2 to 20 ng/ml with an ED50 of about 1.6 ng/ml. These concentrations are well within the physiologic maternal serum free alpha levels which average 350 ng/ml during the third trimester of pregnancy. Incubation of decidual cells with a reference preparation of intact hCG (CR123) at a concentration of 260 ng/ml resulted in stimulated secretion of prolactin, however, the observed stimulation could be attributed to contamination of the preparation with free alpha or dissociated hCG alpha subunit. Purified hCG beta subunit had no stimulatory activity on the decidual cell culture. The effect of alpha subunit on the stimulated release of prolactin was not due to a generalized stimulation of protein synthesis and secretion since no increase was observed in the release of 35S-labeled proteins compared to controls. In addition, the observed increase in prolactin secretion was not due to a toxic effect of the alpha subunit since there was no visible effect on cell viability, and the cellular enzymes, LDH and alkaline phosphatase, were not detected in the culture medium. Addition of exogenous hCG alpha subunit to primary cultures of human trophoblast cultures did not result in stimulated release of human placental lactogen. We conclude that free alpha molecules of pregnancy stimulate release of prolactin from human decidual cells in culture. These results suggest a novel role for free alpha in the paracrine regulation of decidual prolactin secretion.
Asunto(s)
Decidua/metabolismo , Hormonas Glicoproteicas de Subunidad alfa/fisiología , Embarazo/orina , Prolactina/metabolismo , Células Cultivadas , Gonadotropina Coriónica , Medios de Cultivo , Decidua/citología , Densitometría , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Femenino , Hormonas Glicoproteicas de Subunidad alfa/farmacología , Hormonas Glicoproteicas de Subunidad alfa/orina , Humanos , ImmunoblottingRESUMEN
The purpose of the study was to examine the role of N-linked oligosaccharides in preventing combination of free alpha molecules with human chorionic gonadotropin (hCG)-beta subunit to form the intact hormone, hCG. Culture media from JEG cells incubated in the presence or absence of Swainsonine were filtered on Sephadex G-100, and free alpha was identified by radioimmunoassay. Swainsonine interferes with glycosylation by inhibiting alpha-mannosidase II, resulting in formation of hybrid structures. Approximately 50% of the free alpha molecules from Swainsonine-treated cells (Swainsonine pool 2) had an apparent molecular size that was smaller than that of free alpha from control cells. The oligosaccharides of control alpha molecules were resistant to endo-beta-N-acetylglucosaminidase H treatment. In contrast, virtually all of the Swainsonine free alpha molecules contained endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides. Swainsonine free alpha and control free alpha molecules were incubated with an excess of hCG-beta subunit, followed by chromatography on Sephadex G-100. Each fraction was assayed by radioimmunoassay for intact hCG and for alpha. Less than 10% of control free alpha molecules combined with hCG-beta. In contrast, 54% of Swainsonine alpha pool 2 and 40% of Swainsonine alpha pool 1 combined with beta to form hCG. Thus, modulation of N-linked oligosaccharide processing converted free alpha molecules to forms that can combine with hCG-beta. These results indicate that the inability of a substantial portion of control free alpha molecules to combine with hCG-beta is due to the presence of N-linked oligosaccharide structures that interfere with combination.
Asunto(s)
Gonadotropina Coriónica/metabolismo , Hormonas Glicoproteicas de Subunidad alfa/metabolismo , Alcaloides/farmacología , Western Blotting , Carbohidratos/análisis , Gonadotropina Coriónica/química , Gonadotropina Coriónica/ultraestructura , Cromatografía en Gel , Concanavalina A/metabolismo , Hormonas Glicoproteicas de Subunidad alfa/química , Glicoproteínas , Glicosilación/efectos de los fármacos , Hexosaminidasas/farmacología , Humanos , Sustancias Macromoleculares , Unión Proteica , Relación Estructura-Actividad , SwainsoninaRESUMEN
beta-Core is the most abundant hCG-related molecule in pregnancy urine. The structure of beta-core as well as aspects of its metabolic clearance suggest that beta-core is a metabolic fragment of the hCG-beta subunit. The occurrence of beta-core in the urine of patients with a broad spectrum of malignancies imparts an important role to beta-core as a tumor marker. The recent development of antisera with enhanced specificity and sensitivity for beta-core will facilitate further studies on the clinical significance of this molecule.
RESUMEN
The carbohydrate compositions of pregnancy-derived hCG alpha (dissociated from intact hCG) and free alpha-subunit were analyzed using a combination of chemical analysis, lectin affinity chromatography, and glycosidase sensitivity. For direct compositional analysis, parallel samples were hydrolyzed in trifluoroacetic acid and analyzed for sialic acid and neutral sugars without prior derivatization. Separation of the monosaccharides was achieved by HPLC on a Dionex CarboPac column eluted at high pH, and the resolved monosaccharides were quantified by pulsed amperometric detection. The amounts of sugar that were found relative to peptide indicated the presence of two N-linked oligosaccharides per molecule on both hCG alpha and free alpha. Free alpha contained 2.5-fold higher amounts of sialic acid and galactose as well as a higher amount of N-acetylglucosamine than did hCG alpha. Free alpha also contained a 6-fold higher amount of fucose than did hCG alpha (1.2 vs. 0.2 residues of fucose/molecule). Serial fractionation of intact hCG alpha and free alpha molecules by lectin affinity chromatography indicated that virtually all of the hCG alpha-subunits contained at least one Concanavalin-A (Con-A)-binding site, whereas as many as 32% of the free alpha molecules could not bind to Con-A. Chromatography on Lens culinaris (Lch) resulted in 12% binding of hCG alpha and approximately 72% binding of free alpha (80-85% of the Con-A-bound free alpha and 47-48% of the Con-A-nonbound free alpha bound to Lch). Endoglycosidase-H (endo-H) treatment of hCG alpha released a portion of the oligosaccharides. The endo-H-released material appeared to be a monoantennary hybrid based on DEAE-binding properties and carbohydrate composition. In contrast to hCG alpha, free alpha was completely resistant to endo-H treatment. Incubation of endo-H-resistant hCG alpha with glycopeptidase-A resulted in the release of two components, which could be separated into monoantennary and biantennary fractions on the basis of size and charge. The collective data suggest that hCG alpha contains primarily monoantennary hybrid oligosaccharide structures and relatively little fucose. In contrast, free alpha contains primarily multiantennary oligosaccharide structures, and most of the free alpha molecules contain at least one oligosaccharide with fucose attached to the asparagine-linked N-acetylglucosamine residue.
Asunto(s)
Carbohidratos/análisis , Fucosa/análisis , Hormonas Glicoproteicas de Subunidad alfa/análisis , Acetilglucosaminidasa/metabolismo , Metabolismo de los Hidratos de Carbono , Secuencia de Carbohidratos , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Concanavalina A , Femenino , Galactosa/análisis , Glucosamina/análisis , Hormonas Glicoproteicas de Subunidad alfa/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Manosa/análisis , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa , Datos de Secuencia Molecular , Ácido N-Acetilneuramínico , Oligosacáridos/análisis , Embarazo , Ácidos Siálicos/análisisRESUMEN
In addition to human chorionic gonadotrophin (hCG), the urine of pregnant women contains a small molecular weight form of the hCG-beta subunit known as beta-core. Human CG-like material has been described in tissues, serum and urine of normal man, particularly in postmenopausal women. We examined different urine preparations from postmenopausal women to determine whether beta-core-like material, as well as hCG-like material, could be detected. We studied an acetone extract of a pool of 11 litres of postmenopausal urine, three different commercial preparations of human menopausal gonadotrophins and two commercial preparations of 'pure' FSH. After Sephadex G-100 chromatography of these various postmenopausal urine extracts, fractions were assayed using four assay systems to detect hCG, beta-core, LH and FSH immunoreactivities. Human CG immunoreactivity was readily detected in all urinary extracts and it eluted in a position indistinguishable from that of purified hCG. In addition to this hCG-like material, all urinary extracts, except the commercial 'pure' FSH preparations, contained material which reacted in the beta-core radioimmunoassay. This beta-core immunoreactive material eluted from Sephadex G-100 in a position corresponding to that of purified pregnancy-derived beta-core. We conclude that the urine of postmenopausal women contains material resembling the beta-core molecule found in pregnancy urine. The origin of this beta-core-like material remains to be determined, and its presence will have an impact on the application of urinary beta-core as a tumour marker.
Asunto(s)
Gonadotropina Coriónica/orina , Menopausia/orina , Fragmentos de Péptidos/orina , Gonadotropina Coriónica Humana de Subunidad beta , Cromatografía en Gel , Femenino , Hormona Folículo Estimulante/análisis , Humanos , Menotropinas/análisis , Radioinmunoensayo/métodosRESUMEN
We used a highly purified preparation of the naturally occurring core fragment of hCG beta (beta-core) and a new RIA for beta-core to investigate the concentrations and behavior of beta-core in serum and urine. We collected serum and 24-h urine specimens from healthy pregnant women during the first trimester of pregnancy. The concentrations of beta-core in serum were determined by analysis of fractions eluted from Sephadex G-100. Serum concentrations of beta-core immunoreactivity were very low (0.13-1.25 micrograms/L), while large amounts of beta-core were excreted in urine during pregnancy (as much as 4-5 mg/day). Interference with measurement by serum factors did not account for the low levels of beta-core immunoreactivity in pregnancy serum. Based on the known urinary clearance rate of beta-core in healthy nonpregnant subjects, we calculated that urinary clearance of serum beta-core accounts for only about 1% of the beta-core in pregnancy urine. We conclude that during pregnancy, the concentrations of beta-core in plasma are measurable, but extremely low, and that most of the beta-core in urine is derived by mechanisms other than urinary clearance of serum beta-core; most likely, these mechanisms involve nephrogenous production of beta-core from precursor molecules such as hCG and hCG beta.